INTRODUCTION

1. IL-6 AND IL-8 STIMULATION OF BREAST CANCER STEM CELLS CONTRIBUTES TO TRASTUZUMAB RESISTANCE FOLLOWING PTEN DELETION PTEN- HER2+ Control HER2+ PTEN- HER2 PTEN pAkt Hasan Korkaya, Gwangil Kim, April Davis, Ahmed A. Quraishi, Rosemarie D’Angelo, Susan Chung, JumanahSaadeh, Amanda K. Paulson, SulingLiu, Tahra Luther, Shawn Clouthier and Max S. Wicha University of Michigan Comprehensive Cancer Center Tubulin 1 2 3 4 INTRODUCTION RESULTS A A B C We and others have provided strong support for the cancer stem cell (CSC) hypothesis, suggesting that breast cancers are driven by a subpopulation of cells which display stem cell properties. Recent studies also suggest that the CSCs are resistant to chemotherapy and radiation therapy. Although the HER2 targeted therapies (trastuzumab) for treatment of HER2 overexpressing breast cancers have demonstrated significant impact on reducing tumor recurrence, one-third of HER2-positive tumors do not respond to trastuzumab and resistance may develop in patients with chronic exposure. Studies have found that nearly 50% of patients who demonstrate an initial response to trastuzumab relapse within a year. Increasing evidence indicates that this resistance may be associated with loss of PTEN (phosphotase and tensin homolog), the gain of function of somatic mutations of PI3KA or truncation of the extracellular domain of HER2. We recently demonstrated in a set of breast cancer cell lines that trastuzumab-sensitive cell lines show decreases in CSC population, however the CSC population in resistant cell lines is not effected by trastuzumab suggesting a role in resistance. We generated a mouse model for trastuzumab resistance by overexpressing HER2 and deleting PTEN in the MCF7 cell line (MCF7-PTEN-HER2+). PTEN deletion in HER2 expressing cells resulted in increased CSC population leading to increased tumorigenicity and metastasis as compared to cells with only HER2 overexpression (MCF7-HER2+). When we tested these cell lines for trastuzumab response invitro, although the trastuzumab treatment of MCF7-HER2+ cells decreased the CSC population it had no effect in MCF7-PTEN-HER2+ cells. Our in vivo experiments demonstrated that PTEN deletion in HER2 overexpressing cells accelerated tumor growth and development of extensive metastasis from primary tumors, properties not found in parental MCF7 or MCF7-HER2+ cells. Furthermore we found that MCF7-PTEN-HER2+ cells secreted significantly higher cytokines than either MCF7-PTEN- or MCF7-HER2 cells suggesting a synergistic effect. Elevated cytokine expression also attracted inflammatory cells in the tumors generated by MCF7-PTEN-HER2+ cells, but not in tumors from MCF7-PTEN- or MCF7-HER2+ cells. Using these MCF7-PTEN-HER2+ xenografts we examined the efficacy of combination of Akt inhibitor, perifosine with anti-IL-6R antibodies. Furthermore, our results suggest that increased cytokine secretion by MCF7-PTEN-HER2+ tumor cells attracts inflammatory cells that perhaps contribute to the aggressive CSC phenotype by secreting CSC specific cytokines and growth factors as illustrated in Figure 1. B MCF7-HER2+ MCF7-PTEN-HER2+ Figure 3. MCF7-PTEN-HER+ xenograft tumors display denovotrastuzumab resistance while MCF7-HER2 cells are sensitive. (A). Increased tumorigenicity and trastuzumab resistance of MCF7-PTEN-HER+ cells in mouse xenografts are well correlated with (B) expansion of Aldefluor-positive breast CSCs. In contrast, Akt inhibitor was able inhibit Aldefluor-positive cells. Figure 2. Down regulation of PTEN and over-expression of HER2 in breast cancer cells (MCF7-PTEN-HER2+) generates aggressive tumor phenotype by expanding breast CSC population. (A) Western blotting showing the PTEN levels and Akt phosphorylation. Tumors generated by corresponding cells. (B) Increased Aldefluor-positive or CD44+/CD24- CSC population in MCF7-PTEN-HER2+. (C) In vivo imaging of tumor cells expressing luciferase. Primary MCF7-PTEN-HER2+ tumors metastasize to multiple organs including lymph nodes , lung and liver. A B Saline Taxetere Perifosine α-IL-6R Pfs+α-IL-6R A 1 2 1 2 2 1 Before treatment 3 4 3 4 3 4 5 5 5 Adjuvant treatment MCF7-HER2+ MCF7-DsRed MCF7-PTEN-HER2+ • IL-6 • IL-8 • RANTES/CCL5 • Angiogenin • PDGF-B B C Non-metastatic tumor Metastatic tumor Advance treatment Confirmed by ELISA Figure 4. Expression levels of different cytokines in MCF7 parental, HER2+ and PTEN-HER2+ cells. A. Cytokine antibody arrays show increased levels of IL-6, IL-8, RANTES, PDGF-B in MCF7-PTEN-HER2+ cells as compared to parental and MCF7-HER2+ cells. B. Graph confirms relative expression of indicated cytokines quantitated by ELISA in parental MCF7-DsRed, MCF7-HER2, MCF7-PTEN, MCF7-PTEN-HER2 cells. Figure 5. Combination of Aktinhibitor, perifosine with anti-IL-6R antibodies has strong tumor suppression as compared to single treatment in tumors generated from MCF7-PTEN-HER+ cells. A. In vivo imaging of mice with MCF7-PTEN-HER2+ xenografts during the course of adjuvant and advance treatment. Combination of perifosine with anti-IL-6R antibody demonstrated stronger tumor suppression (B) and shows synergistic activity against CSCs in both adjuvant and advance setting in NOD-SCID mice xenografts. CONCLUSION We previously demonstrated that HER2 regulates the breast cancer stem cell population in cells with wild type PTEN. Recent studies demonstrated that resistance to HER2 blocking agents (trastuzumab) have been associated with PTEN deletion in human breast cancer patients. Our studies, demonstrated here, suggest that the resistance is driven by the breast CSC population. PTEN deletion in HER2 overexpressing cells expanded breast CSCs as well as generated an aggressive CSC phenotype which failed to respond to trastuzumab. In order to generate a model of trastuzumab resistant breast cancer, we engineered MCF7 cells with HER2 overexpression, PTEN deletion or the combination (MCF7-PTEN-HER2+). We have found that the combination of PTEN deletion and HER2 overexpression significantly increases the tumor growth in mouse xenograft models and are highly metastatic to multiple organs, a phenotype distinctly different from parental MCF7 cells. Furthermore, by examining cytokine profile expressions, we find that MCF7-PTEN-HER2+ cells secrete several fold more IL-8, IL-6, and CCL5 than parental cells. In addition, combination of Akt inhibitor with IL-6 and IL-8 targeted antibodies suppress tumors generated from MCF7 PTEN-HER2+ cells, strongly suggesting a role for cytokines in aggressive CSC phenotype. These preliminary experiments demonstrate that breast cancer stem cells are regulated by both intrinsic and extrinsic signaling pathways, suggesting that simultaneous blockade of these pathways may prove more efficacious than blocking either pathways alone. CSC Differentiated Cancer Cell Mesenchymal Cells Macrophages Fibroblasts Immune cells Adipocytes Growth Factor Cytokines Figure 1. The tumor microenvironment. Elevated levels of cytokines/growth factors produced by tumor cells enhance the proliferation and survival of cancer stem cells, induces angiogenesis and recruit tumor-associated macrophage, neutrophils and mast cells which secrete additional growth factors forming a positive feedback loop that promotes tumor cell invasion and metastasis.